The antiglobulin test using anti-C3d serum has become a most important tool for the detection of red cell-bound C3d in patients with acquired hemolytic anemias. Graham et al (1976) have reported the presence of C3d on most normal human red cells which may affect proper detection of the cell-bound C3d. We observed (Hsu et al, 1977a) that all normal human red cells were coated with C3d to various degrees, which could be almost completely removed by treatment of the cells with trypsin. In contrast, the cell-bound C3d resulting from complement activation in vitro or in patients with cold agglutinin disease was found to be predominantly trypsin-resistant. Additional data (unpublished) are presented showing the advantage of using trypsinized red cells for C3d antiglobulin tests as well as the limitation of the test. Thus, we propose a simple isotope method to be evaluated for the distinction between the "normal" and "abnormal" C3d on the surfaces of the red cells, and quantitative determination of red cell-bound "abnormal" C3d in patients with acquired hemolytic anemias. The procedures include 1) production of monospecific rabbit anti-human C3d (Hsu et al, 1977b), 2) G-200 fractionation of the anti-C3d serum, 3) labelling the IgG anti-C3d fraction with I125, and 4) the uptake of I125-IgG anti-C3d by trypsinized red cells.